Study on the relationship between heat shock protein 70 and toll-like receptor-4 of monocytes

Abstract

To explore the relation between human heat shock protein 70 (hsp70) and TLR4 in human monocytesin vitro, human monocytes were stimulated with various concentrations of HSP70, and TNF-α production in supernatants was measured by ELISA. Pre-incubated with or without anti-TLR4 mAb, and stimulated with hsp70 (5.0 μg/ml), NF-κB p65 of human monocytes in different time points were detected by immunohistochemistry and monocyte surface expression of TLR4 was measured by flow cytometry. After the human monocytes were pre-incubated with various concentrations of anti-TLR4 and stimulated with hsp70 (5.0 μg/ml), TNF-α production in supernatants was measured. The results showed that hsp70 enhanced NF-κB activation, which was clearly inhibited by anti-TLR4, with the positive cell ratios being 67.44%, 39.17%, 31.56% and 28.05%, respectively. TLR4 was rapidly down-regulated in the presence of hsp70. MFI of TLR4 on monocytes in different time points were 87.77±5.38, 78.16±6.01 and 45.17±4.97 (P<0.05), 26.98±5.83 (P<0.01), respectively. Moreover, hsp70-induced TNF-α production by human monocytes was inhibited by anti-TLR4. It is suggested that TLR4 is involved in the hsp70-mediated activation of innate immunity.

Keywords

heat shock proteins / Toll-like receptors / inflammation / monocyte

References

[1] AseaA, KraeftS K, Kurt-JonesE A, et al. . HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and a cytokine. Nat Med, 2000, 6: 435-435

[2] PeetermansW E, RaatsC J, van FurthR, et al. . Mycobacterial 65-kilodalton heat shock protein induces tumor necrosis factor alpha and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages. Infect Immun, 1995, 63(9): 3454-3454

[3] AkiraS, TakedaK, KaishoT. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol, 2001, 2(8): 675-675

[4] MorisatoD, AndersonK V. The spatzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo. Cell, 1994, 76(4): 677-677

[5] UlevitchR J, TobiasP S. Recognition of gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol, 1999, 11(1): 19-19

[6] ChuangT, UlevitchR J. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta, 2001, 1518(1–2): 157-157

[7] OhtsukaK, HataM. Molecular chaperone function of mammalian Hsp70 and Hsp40-a review. Int J Hyperthermia, 2000, 16(3): 231-231

[8] SrivastavaP K, MenoretA, BasuS, et al. . Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity, 1998, 8(6): 657-657

[9] SondermannH, BeckerT, MayhewM, et al. . Characterization of a receptor for heat shock protein 70 on macrophages and monocytes. Biol Chem, 2000, 381(12): 1165-1165

[10] TriantafilouM, MiyakeK, GolenbockD T, et al. . Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J Cell Sci, 2002, 15: 2603-2603

[11] VolkH D, ReinkeP, DockeW D. Clinical aspects: from systemic inflammation to immunoparalysis. Chem Immunol, 2000, 74: 162-162